Two genes specifically expressed in fruiting dikaryons of Schizophyllum commune: homologies with a gene not regulated by mating-type genes

The nucleotide (nt) sequences of the Sc3 and Sc4 genes of the filamentous fungus Schizophyllum commune, and the deduced amino acid (aa) sequences, were determined; moreover, the previously published sequence for the ScI gene [Dons et al., EMBO J. 3 (1984) 2101–2106] was corrected. All three independently isolated genes were found to have similar structures and nt sequences of their coding regions. At the aa level the homology is 43–62% (63–69% in the C-terminal parts of the proteins), the hydrophobic aa predominate and the hydrophobicity patterns are similar. All three proteins contain leader sequences and eight cysteines among about 110 aa, conserved at the same positions. Yet these genes are differentially regulated: Sc1 and Sc4 are only expressed at high levels in fruiting dikaryons, whereas Sc3 is highly expressed in both monokaryons and dikaryons, independent from fruiting.     

Characterization and expression of three novel differentiation-related genes belong to the human NDRG gene family

NDRG1(N-Myc downstream regulated) is upregulated during cell differentiation, repressed by N-myc and c-myc in embryonic cells, and suppressed in several tumor cells. A nonsense mutation in the NDRG1 gene has been reported to be causative for hereditary motor and sensory neuropathy-Lom (HMSNL), indicating that NDRG1 functions in the peripheral nervous system necessary for axonal survival. Here, we cloned three human cDNAs encoding NDRG2 (371aa), NDRG3 (375aa) and NDRG4 (339aa), which are homologous to NDRG1. These three genes, together with NDRG1, constitute the NDRG gene family. The phylogenetic analysis of the family demonstrated that human NDRG1 and NDRG3 belong to a subfamily, and NDRG2 and NDRG4 to another. At amino acid (aa) level, the four members share 53–65% identity. Each of the four proteins contains an / hydrolase fold as in human lysosomal acid lipase. Expression of the fusion proteins NDRG2/GFP, NDRG3/GFP and NDRG4/GFP in COS-7 cells showed that all of them are cytosolic proteins. Based on UniGene cluster analysis, the genes NDRG2, NDRG3 and NDRG4 are located at chromosome 14q11.1–11.2, 20q12–11.23 and 16q21–22.1, respectively. Northern and dot blot analysis shows that all of the three genes are highly expressed in adult brain and almost not detected in the eight human cancer lines. In addition, in contrast to the relatively ubiquitous expression of NDRG1, NDRG2 is highly expressed in adult skeletal muscle and brain, NDRG3 highly expressed in brain and testis, and NDRG4 specifically expressed in brain and heart, suggesting that they might display different specific functions in distinct tissues.     

Argininosuccinate synthetase and argininosuccinate lyase: two ornithine cycle enzymes from Agaricus bisporus

Accumulation of high quantities of urea in fruiting bodies is a known feature of larger basidiomycetes. Argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) are two ornithine cycle enzymes catalysing the last two steps in the arginine biosynthetic pathway. Arginine is the main precursor for urea formation. In this work the nucleotide sequences of the genes and corresponding cDNAs encoding argininosuccinate synthetase (ass) and argininosuccinate lyase (asl) from Agaricus bisporus were determined. Eight and six introns were present in the ass and asl gene, respectively. The location of four introns in the asl gene were conserved among vertebrate asl genes. Deduced amino acid sequences, representing the first homobasidiomycete ASS and ASL protein sequences, were analysed and compared with their counterparts in other organisms. The ass ORF encoded for a protein of 425 amino acids with a calculated molecular mass of 47 266 Da. An alignment with ASS proteins from other organisms revealed high similarity with fungal and mammalian ASS proteins, 61–63 % and 51–55 % identity, respectively. The asl open reading frame (ORF) encoded a protein of 464 amino acids with an calculated mass of 52 337 Da and similar to ASS shared the highest similarity with fungal ASL proteins, 59–60 % identity.Northern analyses of ass and asl during fruiting body formation and post-harvest development revealed that expression was significantly up-regulated from developmental stage 3 on for all the tissues studied. The expression reached a maximum at the later stages of fruiting body growth, stages 6 and 7. Both ass and asl genes were up-regulated within 3 h after harvest showing that the induction mechanism is very sensitive to the harvest event and emphasizes the importance of the arginine biosynthetic pathway/ornithine cycle in post-harvest physiology.     

Plasmodium falciparum: recombinant baculoviruses direct the expression of circumsporozoite proteins in Spodoptera frugiperda cell cultures

The DNA coding for the circumsporozoite protein (CPS) of Plasmodium falciparum has been cloned into the baculovirus expression vector pAcYM1 and expressed in Spodoptera frugiperda (Sf9) insect cells. Three DNA constructs have been made: the first one directs the synthesis of the complete CSP (aa 1–412), the second leads to the production of a species devoid of the anchor domain (aa 1–391) and the third one to a molecule lacking both signal and membrane anchor sequences (aa 18–391). All three recombinant CPS were produced at about 3 g per 10⁶ infected cells and were characterized in terms of immunoreactivity and apparent molecular weight. Analytical purification of the recombinant proteins was achieved by a combination of heat treatment, acidification, isoelectric focusing and ion exchange chromatography. The purified material, when injected into mice, generated only modest antibody responses, although antisera from immunized mice reacted with control CSP antigens carrying or not the major immunodominant repeat region.Abbreviations AcNPV Autographa californica nuclear polyhedrosis virus - CSP circumsporozoite protein     

In Arabidopsis thaliana, 1% of the genome codes for a novel protein family unique to plants

In the sequences released by the Arabidopsis Genome Initiative (AGI), we discovered a new and unexpectedly large family of orphan genes (127 genes by 01.08.99), named AtPCMP. The distribution of the AtPCMP genes on the five chromosomes suggests that the genome of Arabidopsis thaliana contains more than 200 genes of this family (1% of the whole genome). The deduced AtPCMP proteins are characterized by a surprising combinatorial organization of sequence motifs. The amino-terminal domain is made of a succession of three conserved motifs which generate an important diversity. These proteins are classified into three subfamilies based on the length and nature of their carboxy-terminal domain constituted by 1–6 motifs. All the motifs characterized have an important level of conservation in both sequence and spacing. A specific signature of this large family is defined. The presence of ESTs in databases and the detection of clones in A. thaliana cDNA libraries indicate that most of the genes of this family are expressed. The absence of similar sequences outside the plant kingdom strongly suggests that this unusually large orphan family is unique to plants. Features, the genesis, the potential function and the evolution of this plant combinatorial and modular protein family are discussed.     

The Cytochrome b5 Tail Anchors and Stabilizes Subdomains of Human DNA Topoisomerase IIα in the Cytoplasm of Retrovirally Infected Mammalian Cells

DNA topoisomerase II (topo II) is the target of many anticancer drugs and is often altered in drug-resistant cell lines. In some tumor cell lines truncated isoforms of topo IIα are localized to the cytoplasm. To study the localization and function of individual enzyme domains, we have epitope-tagged several fragments of human topo IIα and expressed them by retroviral infection of rodent and human cells. We find that fusion of the topo II fragments to the hydrophobic tail of human liver cytochrome b₅ anchors the fusion protein to the outer face of cytoplasmic membranes, as determined by colocalization with calnexin and selective detergent permeabilization. Moreover, whereas the minimal ATPase domain (aa 1–266) is weakly and diffusely expressed, addition of the cytb₅ anchor (1–266-b₅) increases its steady-state level 16-fold with no apparent toxicity. Similar results are obtained with the complete ATPase domain (aa 1–426). A C-terminal domain (aa 1030–1504) of human topo IIα containing an intact dimerization motif is stably expressed and accumulates in the nucleus. Fusion to the cytb₅ anchor counteracts the nuclear localization signal and relocalizes the protein to cytoplasmic membranes. In conclusion, we describe a technique that stabilizes and targets retrovirally expressed proteins such that they are exposed on the cytoplasmic surface of cellular membranes. This approach may be of general use for regulating the nuclear accumulation of drugs or proteins in living cells.     

A new subtilisin family: nucleotide and deduced amino acid sequences of new high-molecular-mass alkaline proteases from<Emphasis Type="Italic"> Bacillus</Emphasis> spp.

Six genes encoding high-molecular-mass subtilisins (HMSs) of alkaliphilic Bacillus spp. were cloned and sequenced. Their open reading frames of 2,394–2,424 bp encoded prosubtilisins of 798–808 amino acids (aa) consisting of the prepropeptides of 151–158 aa and the mature enzymes of 640–656 aa. The deduced aa sequences of the mature enzymes exhibited 60–95% identity to those of FT protease of Bacillus sp. strain KSM-KP43, a subtilisin-like serine protease, and a minor serine protease, Vpr, of Bacillus strains. Three of the six recombinant enzymes were susceptible to proteolysis, but the others were autodigestion resistant. All enzymes had optimal pH values of 10.5–11.0, optimal temperatures of 40–45°C for hydrolysis of a synthetic substrate, and were heat labile. These alkaline proteases seem to form a new subtilisin family, as judged by their aa sequences and phylogenetic analysis.Communicated by K. Horikoshi     

Characterization of a Gy4 glycinin gene from soybean Glycine max cv. Forrest

The glycinin gene family encoding the glycinin subunits in soybean plants is composed of at least five gene members. A genomic clone S312 containing the Gy4 gene from a genomic library of cv. Forrest was isolated and partially characterized. The organization of this gene was found to be similar to that of a null allele from cv. Raiden, but different from the Gy4 gene from cv. Dare. The complete nucleotide sequence of this gene has been determined. It is 2599 bp long consisting of four exons and three introns. Comparing the DNA sequences between this gene and the gene from Dare and a null allele from Raiden, the difference found in the coding region was 5-GCAGTGCAAG-3 (nt 824 to 833) in the former case versus 5-TGGAGTTGCAATT-3 (nt 1314 to 1326) in the latter case in the exon 2 domain, resulting in three amino acid differences and one amino acid absence. Some other differences were also found in the non-coding region. The coding sequence and 5-flanking region of the Gy4 gene, when compared with that of other legumin genes as well as group 1 glycinin subunit genes, revealed some interesting features: (1) a transposable element-like sequence was found in the hypervariable region (HVR) of the exon 3 domain, which was lacking in the legumin and the glycinin group 1 genes; (2) in the 5-flanking region from nt –145 to –1, two high-homology sequences were found: one from nt –141 to nt –132, the other from nt –118 to nt –92 which includes the legumin box and the RY repeat element.     

Domain organization and intron positions inCaenorhabditis elegans collagen genes: The 54-bp module hypothesis revisited

Summary The amino acid (aa) sequences of the polypeptides encoded by five collagen genes of the nematodeCaenorhabditis elegans, col-6, col-7 (partial),col-8, col-14, andcol-19, were determined. These collagen polypeptides, as well as those encoded by the previously sequencedC. elegans collagen genescol-1 andcol-2, share a common organization into five domains: an amino-terminal leader, a short (30–33 aa) (Gly-X-Y) _n domain, a non(Gly-X-Y) spacer, a long (127–132 aa) (Gly-X-Y) _n domain, and a short carboyl-terminal domain. The domain organizations and intron positions of these polypeptides were compared with those of the polypeptides encoded byDrosophila andStrongylocentrotus type IV, and vertebrate types I, II, III, IV, and IX collagen genes; theC. elegans collagen polypeptides are most similar to the vertebrate type IX collagents. It is suggested that the collagen gene family comprises two divergent subfamilies, one of which includes the vertebrate interstitial collagen genes, and the other of which includes the invertebrate collagen genes and the vertebrate type IV and type IX collagen genes. Only the vertebrate interstitial collagen genes display clear evidence of evolution via the tandem duplication of a 54-bp exon.     

The sequence of a porcine cDNA encoding α-lactalbumin

A cDNA clone encoding porcine α-lactalbumin (αLA) was isolated and sequenced. The longest clone was 688 nucleotides (nt) long and encoded a preprotein of 141 amino acids (aa) including a leader peptide of 19 aa. The porcine cDNA exhibited a nt similarity of between 72.2%–83.5% to other αLA cDNAs and an aa similarity of between 50.8%–85.2% with other αLA aa sequences. The derived aa sequence varied at three positions from a previously reported sequence for porcine αLA obtained by direct aa sequencing.     

Expression of A mating type genes of Coprinus cinereus in a heterologous basidiomycete host

The A mating factor of Coprinus cinereus determines compatibility in mating by regulating part of a developmental sequence that leads to dikaryon formation. The A genes that trigger development encode two different classes of homeodomain proteins, and for a successful mating, a protein of one class, HD 1, must interact with a protein of the other class, HD 2. In this report we show that C. cinereus A genes that encode HD 2 proteins, a2-1 and b2-1, can elicit A-regulated development in the heterologous host C. bilanatus. Transformation rates were very low, suggesting that the genes were poorly transcribed. The fact that the HD 2 genes are functionally expressed implies successful heteromultimeric association of putative DNA-binding proteins coded by the two Coprinus species. This interaction was sufficient to satisfy the need for different A factors in the formation of a fertile C. bilanatus dikaryon, but fertile dikaryons were more readily produced in matings with the a2-1 gene transformants. The C. cinereus A genes, b1-1 and d1-1, which encode HD1 proteins, were either not expressed or their proteins were non-functional in C. bilanatus. These experiments raise some interesting questions regarding HD1–HD2 protein interactions.     

Structure of the three beta-tubulin-encoding genes of the unicellular alga, Polytomella agilis

The quadriflagellate, unicellular, colorless alga, Polytomella agilis, contains several distinct microtubule arrays. To study the genetic basis of microtubule heterogeneity in P. agilis, we characterized its tubulin(Tub)-encoding genes (tub). The three beta tub genes detected in blots of P. agilis DNA were isolated from a genomic library. The structure and organization of the genes were examined by restriction mapping and nucleotide (nt) sequencing. S1 nuclease protection studies showed that all three genes are expressed. The predicted amino acid (aa) sequences are more than 98% conserved with the Chlamydomonas reinhardtii and Volvox carteri beta-Tubs, underscoring the close phylogenetic relationship of these species. Evolutionary divergence among the P. agilis genes is demonstrated by differences in intron number, nt sequences in noncoding regions, and silent nt substitutions in the coding regions. However, the proteins encoded by the beta 1 and beta 3 tub genes are identical; the beta 2 gene product differs by one conservative aa substitution. These results are in striking contrast to the C-terminal aa diversity reported within beta tub gene families in animal, higher plant and fungal systems. The data support the hypothesis that those tub genes whose products assemble into axonemal microtubules are subject     

Analysis of genes expressed in head kidney of common carp Cyprinus carpio L treated with cortisol

We analyzed genes expressed from head kidney of common carp Cyprinus carpio L. treated with cortisol. The results of single-pass sequencing of expressed sequence tags (ESTs) from 188 clones (AU240288–AU240367, AU301120–AU301227) from kidney cDNA are presented. One-hundred-twenty-seven clones (67.6%) were completely unknown and are likely to represent newly described genes, whereas 61 clones (32.4%) were identified based on matches to sequences in the database. The putative genes contain several ribosomal proteins, cytochrome oxidase subunits. Immune related cDNA clones identified from kidney were immunoglobulin light chain (n=4), FK506/rapamycin-binding protein (FKBP), CXC chemokine receptor type 4, complement factor B/C2-A3, peptidylprolyl isomerase (cyclophilin; Cyp)-like1, cyclophilin S1, heat shock-70 kDa protein-4, stress-activated protein kinase-3 (n=2). FKBP and cyclophilin genes expressed in normal tissues (head kidney, spleen, liver, brain and heart). Expression of FKBP and cyclophilin genes were not detected in liver, brain and heart when treated with cortisol for 16 h.     

A unigene catalogue of 5700 expressed genes in cassava

Two economically important characters, starch content and cassava bacterial blight resistance, were targeted to generate a large collection of cassava ESTs. Two libraries were constructed from cassava root tissues of varieties with high and low starch contents. Other libraries were constructed from plant tissues challenged by the pathogen Xanthomonas axonopodis pv.manihotis. We report here the single pass sequencing of 11 954 cDNA clones from the 5’ ends, including 111 from the 3’ ends. Cluster analysis permitted the identification of a unigene set of 5700 sequences. Sequence analyses permitted the assignment of a putative functional category for 37% of sequences whereas ~ 16% sequences did not show any significant similarity with other proteins present in the database and therefore can be considered as cassava specific genes. A group of genes belonging to a large multigene family was identified. We characterize a set of genes detected only in infected libraries putatively involved in the defense response to pathogen infection. By comparing two libraries obtained from cultivars contrasting in their starch content a group of genes associated to starch biosynthesis and differentially expressed was identified. This is the first large cassava EST resource developed today and publicly available thus making a significant contribution to genomic knowledge of cassava.     

Two Novel Human Members of an Emerging Mammalian Gene Family Related to Mono-ADP-Ribosylating Bacterial Toxins

Mono-ADP-ribosylation is one of the posttranslational protein modifications regulating cellular metabolism, e.g., nitrogen fixation, in prokaryotes. Several bacterial toxins mono-ADP-ribosylate and inactivate specific proteins in their animal hosts. Recently, two mammalian GPI-anchored cell surface enzymes with similar activities were cloned (designated ART1 and ART2). We have now identified six related expressed sequence tags (ESTs) in the public database and cloned the two novel human genes from which these are derived (designatedART3andART4). The deduced amino acid sequences of the predicted gene products show 28% sequence identity to one another and 32–41% identity vs the muscle and T cell enzymes. They contain signal peptide sequences characteristic of GPI anchorage. Southern Zoo blot analyses suggest the presence of related genes in other mammalian species. By PCR screening of somatic cell hybrids and byin situhybridization, we have mapped the two genes to human chromosomes 4p14–p15.1 and 12q13.2–q13.3. Northern blot analyses show that these genes are specifically expressed in testis and spleen, respectively. Comparison of genomic and cDNA sequences reveals a conserved exon/intron structure, with an unusually large exon encoding the predicted mature membrane proteins. Secondary structure prediction analyses indicate conserved motifs and amino acid residues consistent with a common ancestry of this emerging mammalian enzyme family and bacterial mono(ADP-ribosyl)transferases. It is possible that the four human gene family members identified so far represent the “tip of an iceberg,” i.e., a larger family of enzymes that influences the function of target proteins via mono-ADP-ribosylation.     

Comprehensive cloning of Schizosaccharomyces pombe genes encoding translation elongation factors

In the course of the Schizosaccharomyces pombe cDNA project, we succeeded in cloning all the genes encoding translation elongation factors EF-1α, EF-1β, EF-1γ, EF-2 and EF-3. With the exception of the EF-1γ gene, the nucleotide (nt) sequence of S. pombe elongation factors has not been previously reported. For EF-1α, we found three genes whose amino acid (aa) sequences are quite homologous each other (99.5%), but whose 3′ untranslated regions (UTRs) are completely different. Southern blot indicated that those three EF-1α genes are located at different loci. Northern analysis indicated that one of three EF-1α genes was inducible with UV-irradiation, while the level of expression for another of three EF-1α genes was repressed by UV and heat-shock (HS) treatments. The aa sequence predicted from the nt sequence of the S. pombe EF-1β cDNA clone covered almost all the coding sequence (CDS) of EF-1β except the first methionine which has 55.4% identity with that of S. cerevisiae. We also identified two copies of S. pombe EF-2 genes. Their aa sequences deduced from nt sequences are identical (100%), but they have different 3′ UTRs. The location of these two EF-2 genes in different loci was proved by Southern analysis. The S. pombe EF-3 cDNA clone encoded only a third of the CDS from the C-terminal and its deduced aa sequence has a 76% identity with those of other yeasts and fungi.